title: Gene expression profiling of individual hypothalamic nuclei from single animals using laser capture microdissection and microarrays.
abstract: In order to identify novel genes involved in appetite and body weight regulation we have developed a microarray based method suitable for detecting small changes in gene expression in discrete groups of hypothalamic neurons. The method is based on a combination of stereological sampling, laser capture microdissection (LCM), PCR based amplification (SuperAmp), and one-color cDNA microarray analysis. To validate the method we assessed and compared fasting induced changes in mRNA levels of Neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the hypothalamic arcuate nucleus (ARC) of diet-induced obese rats using cDNA microarrays, quantitative PCR and in situ hybridization. All methods revealed statistically significant fasting-induced changes in NPY and POMC expression. An additional 3480 differentially expressed probes (fold change >1.22, t-test p=0.05) were identified in the microarray analysis. Our findings demonstrate a consistent gene expression pattern across three different gene expression detection methods and strongly suggest that LCM coupled microarray analysis combined with SuperAmp can be used as a semi-quantitative mRNA profiling tool. Importantly, the sensitivity of the method greatly improves the usefulness of the microarray technology for gene expression profiling in non-homogeneous tissues such as the brain.
authors.affiliation: Rheoscience A/S, Glerupvej 1, DK-2610 Rødovre, Denmark; University of Southern Denmark, BMB, Campusvej 55, DK-5230 Odense M, Denmark. sjp@rheoscience.com
authors.forename: Sarah Juel
authors.initials: SJ
authors.lastname: Paulsen
authors.forename: Leif Kongskov
authors.initials: LK
authors.lastname: Larsen
authors.forename: Jacob
authors.initials: J
authors.lastname: Jelsing
authors.forename: Uwe
authors.initials: U
authors.lastname: Janssen
authors.forename: Bernhard
authors.initials: B
authors.lastname: Gerstmayer
authors.forename: Niels
authors.initials: N
authors.lastname: Vrang
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001111:Arcuate Nucleus of Hypothalamus / Q000378:metabolism*; D004041:Dietary Fats / Q000009:adverse effects; D005215:Fasting / Q000502:physiology; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology*; D008297:Male; D046228:Microarray Analysis / Q000379:methods*; D042282:Microdissection / Q000379:methods*; D009478:Neuropeptide Y / Q000235:genetics / Q000378:metabolism; D009765:Obesity / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D011333:Pro-Opiomelanocortin / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D004041:Dietary Fats; D009478:Neuropeptide Y; D012333:RNA, Messenger; D011333:Pro-Opiomelanocortin